Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization.

Different bacterial isolates with amylolytic activity were insulated from various honey samples. The most active isolate was identified by the molecular 16SrRNA sequence technique as Bacillus atrophaeus NRC1. The bacterium showed maximum amylase production under optimum culture conditions at pH 6.0, 40 °C and after 24 h incubation. Two amylase isoenzymes (AmyI and AmyII) from Bacillus atrophaeus NRC1 have been purified to homogeneity by using ammonium sulfate precipitation, Sephacryl S-200 and DEAE-Sepharose chromatography. The major isoenzyme, AmyI, had a specific activity 4635 U/mg proteins with molecular weight of 61 kDa using SDS-PAGE electrophoresis. The maximum activity of AmyI against starch was determined at pH 6.0 and 50 °C. AmyI was stable up to 50 °C after incubation for 30 min, retained 65 and 23% of its activity at 60 and 70 °C, respectively. Pre-incubation with Ca2+, Mg2+ and Ba2+ cations for 30 min enhanced the enzyme activity; while it was completely inhibited by Hg2+. Varied inhibition degree of the enzyme activity was determined with K+, Ni2+, Zn2+, Na2+ and Cu2+ ions. AmyI was inhibited by EDTA, PMSF and SDS, while it was activated by l-Cysteine-HCl and DTT. AmyI had the ability to degrade starch, amylopectin, glycogen, amylose and lacked the affinity towards ß-1,4-linked xyloses.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 24 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes.

Production, immobilization and thermodynamic studies of free and immobilized Aspergillus awamori amylase.

Enzyme cost, stability and its thermodynamic characteristics are the main criteria for industrial use. In this study, Aspergillus awamori amylase was constitutively produced using various agro-industrial wastes. Olive oil cake gave the highest activity (230U/g). The amylase was partially purified to 2.81-fold purification. Immobilization was achieved using different carriers by covalent binding. The novel carrier Ca+2 alginate (Alg) starch (St)/polyethyleneimine (PEI)/glutaraldehyde (GA), showed the highest operational stability and was selected for further studies. The optimum temperature for the free and immobilized form was 50°C and 55-60°C, respectively. The immobilization process had a major role in improving enzyme thermal stability. In comparison to free enzyme, the immobilized form showed the highest optimum temperature, activation energy (Ea) and deactivation rate constants (kd). Also, t1/2, D-values (decimal reduction time), change in enthalpy (ΔH° kJmol-1), and Gibbs free energy (ΔG°) increased and was higher than the native enzyme within 50-80°C. The magnitude of negative value of entropy (ΔS° kJmol-1) for immobilized enzyme was negative for the free and immobilized enzymes revealing that native form of enzyme was in more ordered state. Km and Vmax values were slightly affected by the temperature variations 40-70°C.

Synthesis of novel 2-(substituted amino)alkylthiopyrimidin-4(3H)-ones as potential antimicrobial agents.

5-Alkyl-6-(substituted benzyl)-2-thiouracils 3a,c were reacted with (2-chloroethyl) diethylamine hydrochloride to afford the corresponding 2-(2-diethylamino)ethylthiopyrimidin- 4(3H)-ones 4a,b. Reaction of 3a-c with N-(2-chloroethyl)pyrrolidine hydrochloride and/or N-(2-chloroethyl)piperidine hydrochloride gave the corresponding 2-[2-(pyrrolidin-1-yl)ethyl]-thiopyrimidin-4(3H)-ones 5a-c and 2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-4(3H)-ones 6a,b, respectively. Treatment of 3a-d with N-(2-chloroethyl)morpholine hydrochloride under the same reaction conditions formed the corresponding 2-[2-(morpholin-4-yl)ethyl]thiopyrimidines 6c-f. On the other hand, 3a,b were reacted with N-(2-bromoethyl)phthalimide and/or N-(3-bromopropyl)phthalimide to furnish the corresponding 2-[2-(N-phthalimido)ethyl]-pyrimidines 7a,b and 2-[3-(N-phthalimido)-propyl]pyrimidines 7c,d, respectively. Compounds 3a-d, 4a,b, 5a-c, 6a-f and 7a-d were screened against Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis NRRL 4219 and Bacillus cereus), yeast-like pathogenic fungus (Candida albicans ATCC 10231) and a fungus (Aspergillusniger NRRL 599). The best antibacterial activity was displayed by compounds 3a, 3b, 4a, 5a, 5b, 6d, 6f, 7b and 7d, whereas compounds 4b, 5b, 5c, 6a, 6b and 6f exhibited the best antifungal activity.

Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.